Method for preparing tissue repair promoting substances

ABSTRACT

A method for isolating tissue repair promoting substances from human or animal blood, which method comprises collecting the human or animal blood from a single human or animal individual in a first container of a container system comprising at least first and second interconnected containers; centrifuging the container system containing said blood so as to separate the blood in various fractions including a plasma fraction; transferring at least part of the plasma fraction to said second container of the container system; subjecting the plasma fraction in said second container to a low temperature so as to obtain a precipitate comprising tissue repair promoting substances; concentrating said precipitate in said second container so as to obtain a first fraction comprising a major part of the non-precipitated material, and a second fraction comprising at least the major part of the precipitate and a minor part of the non-precipitated material; and separating said second fraction comprising the tissue repair promoting substances from said first fraction within the second container; and optionally heating said second fraction within one of the containers of the container system so as to dissolve the isolated precipitated tissue repair promoting substances. 
     Furthermore, fractions comprising tissue repair promoting substances derived from blood cells may be isolated within the container system, said fractions optionally being combined with the second fraction comprising tissue repair promoting substances derived from blood plasma. 
     At least part of the fractions obtained may be kept for application to a tissue of the human or animal individual from whom the blood was collected.

This is a continuation of application Ser. No. 07/704,911, filed May 21,1991 now abandoned which was a continuation of application Ser. No.07/465,430 filed Jan. 17, 1990, now abandoned, which was a continuationof application Ser. No. 07/216,712, filed Jul. 5, 1988, now abandoned.

FIELD OF THE INVENTION

The present invention provides a simple, rapid, and inexpensive methodand apparatus for small-scale preparation of tissue repair promotingsubstances needed in medical and veterinary practice. The substances areisolated from biological material obtained from human or animalindividuals. The compositions comprising the biologically activesubstances prepared according to the invention offer significantbenefits to the subject treated therewith in that they, in addition ofefficiently promoting tissue repair, have few or no harmful sideeffects.

BACKGROUND OF THE INVENTION

In recent years several naturally occurring biological substances,especially from mammals, have been isolated and their biologicalproperties have been established. Parallel to this development, fieldsof application of these substances have been discovered, and the need ofsuitable methods for their isolation have therefore increased. Examplesof such substances are substances recovered from human blood, andmethods for large-scale production using blood pooled from severaldonors have been developed.

However, the drawbacks of these large-scale production methods arenumerous. First, the obtainment of blood from donors (voluntary or paid)causes well-known problems, such as the problem of limited access to asufficient amount of biological material due to limitations with respectto the number of donors and the amount of blood which can be collectedfrom the individual donor. Secondly, the collection procedures, storageand distribution of the material obtained require time and considerablefinancial ressources. Thirdly, the large-scale production of thesubstances under sterile conditions necessitates advanced and expensiveequipment, and the waste of material during these numerous and laboriousisolation steps is substantial.

Furthermore, the quality of the substances in the product obtained issubstantially decreased compared to the quality of the substancespresent in the original native biological material. Thus, the yield ofbiologically active well-functioning substances may be very low. Storageof biological material over prolonged periods is known to result inspontaneous degradation of the various substances. Procedures (i.e.heating) employed in order to prevent/reduce the risk of transmission ofinfectious microorganisms substantially increase the extent of thedenaturation of the substances and thus the loss of biological activity.A method which completely inactivates any kind and any amount ofmicroorganisms and at the same time preserves the biological activity ofthe substances in question has not yet been developed.

A product prepared from numerous donors is inhomogeneous, and everycomponent in the product represents a potential immunogen, i.e.administering the product to a human or animal individual in needthereof may result in harmful immunological reactions in saidindividual. The severity of such allergic/immunological reactions rangesfrom minor inconveniences to fatal anaphylactic reactions or disablingimmunological diseases.

A field in which the administration of biological substances has becomeimportant is the field of promoting tissue repair, e.g. wound healing.It is now known that several substances contribute to the obtainment oftissue repair. During the repair of a tissue injury, chain reactionswhich release several substances occur, and these substances contributeto the body's mechanisms of tissue repair including hemostasis.Essential to this complex defence mechanism of the body are substancesnaturally present in whole blood. Some of these substances are presentin plasma under normal conditions and undergo modifications during thetissue repair process. Other substances are under normal conditionspresent in the blood cells and are released from these cells during thetissue repair process.

Coagulation factors are substances in the blood that are essential tothe clotting process and hence, to the maintenance of the hemostasisneeded in tissue repair. Blood coagulation is the sequential process bywhich the multiple coagulation factors of the blood interact, ultimatelyresulting in an insoluble fibrin network. Coagulation factor I is alsocalled fibrinogen. Fibrinogen can be converted into fibrin which formsthe essential part of the blood clot. Fibrin is converted to aninsoluble supporting network of fibrous material. The conversion takesplace by the action of an enzyme such as thrombin. Coagulation factorVIII, also known as the antihemophilic factor, participates in bloodcoagulation. Coagulation factor XIII is the fibrin stabilizing factor.It polymerizes fibrin monomers so that they become stable and insoluble,thus enabling the formation of a stable clot.

Also participating in the coagulation processes are the platelet derivedfactors, the so-called platelet factors 1-4, e.g. platelet factor 2,which accelerates the thrombin-fibrinogen reaction. The platelet-derivedgrowth factor (PDGF) is known as a potent mitogen, which e.g. promotesthe proliferation of fibroblast cells needed in tissue repair.Furthermore, epidermal growth factor (EGF) which promotes epithelialgrowth is present in the platelets.

Another important component present in plasma is the proteinfibronectin, which plays a role in cell proliferation and in fibrininteraction.

Knowledge of the molecular basis for the sequence of the biochemicalphenomena involved in tissue repair has promoted the need fordevelopment of suitable isolation procedures for the various biochemicalsubstances involved in the repair. Compositions comprising the isolatedtissue repair promoting substances have many applications in medical andveterinary practice, e.g. in wound repair and in oozing surfaces, ascontrol of local bleeding, e.g. in liver and spleen repair, inprevention of peritoneal adhesion-formation by local fibrin sealing, asclosure of postoperative fistulae, and as fixation of skin grafts. Theapplication of a composition containing tissue repair promotingsubstances in a concentrated form to a mammal in need of the treatment,e.g. treatment of a surgical wound, may shorten the wound healing periodconsiderably.

An important aspect of the use and manufacture of compositionscomprising tissue repair promoting substances is the employment of thecomposition for achieving substantial intermediary initial adhesionbetween injured tissue surfaces. E.g. wound healing can be efficientlypromoted by topically applying a composition comprising fibrinogen in aconcentrated form to a mammal in need of the treatment, and at the sametime providing means for the enzymatic conversion of the fibrinogen to afirm supporting fibrous network. The use of such a tissue-adhesivesystem will probably increase, especially in surgical practice where itprovides excellent means for obtaining hemostasis, closing leakages fromfluid containing compartments etc.

The isolation and use of fibrinogen compounds for tissue repair iswell-known, cf. AT No. 359,653, DE No. 3,002,934, DE No. 3,203,775, DENo. 3,105,624, EP No. 0,068,047, WO 86/01814 and EP No. 0,068,149.

These known methods are typically large-scale preparations of substancesfrom a blood pool obtained from more than one donor, often from a bloodpooled from several hundreds or thousands of individual donors.Furthermore, the procedures usually involve several laboriouspurification steps such as repeated washing procedures. Usuallylyophilization, which is a laborious time- and material-consumingprocedure, is employed. Sterilization procedures such as sterilefiltration are often needed. The overall recoveries of biologicallyactive substances recovered by such procedures are low.

SUMMARY OF THE INVENTION

The object of the present invention is the provision of a simple andinexpensive method for small-scale production of substances useful intissue repair, in particular substances isolated from blood plasma,e.g., coagulation factors, and substances derived from blood cells, suchas the platelet-derived growth factor (PDGF).

The present invention provides a method for the production of acomposition comprising tissue repair promoting substances such as thecoagulation factors fibrinogen and factor XIII, and the adhesive proteinfibronectin. Furthermore, the composition may comprise tissue repairpromoting substances derived from blood cells such as leucocytes orplatelets, e.g. the platelet-derived growth factor (PDGF) and epidermalgrowth factor (EGF).

The invention further provides an apparatus or system for thepreparation of tissue repair promoting substances, said apparatuspermitting the preparation to be performed rapidly and with minimum useof expensive advanced equipment. The apparatus is small (the overallweight of the apparatus typically being less than one kg), i.e. it caneasily be transported to the donor of the biological material, sealedfrom the environment, and subsequently easily transported to alaboratory having conventional equipment such as a centrifuge and acooling bath.

In its broadest aspect, the method of the invention comprises

a) collecting the human or animal blood from a single human or animalindividual in a first container of a container system comprising atleast first and second interconnected containers,

b) centrifuging the container system containing said blood so as toseparate the blood in various fractions including a plasma fraction,

c) transferring at least part of the plasma fraction to said secondcontainer of the container system,

d) subjecting the plasma fraction in said second container to a lowtemperature so as to obtain a precipitate comprising tissue repairpromoting substances.

e) concentrating said precipitate in said second container so as toobtain a first fraction comprising a major part of the non-precipitatedmaterial, and a second fraction comprising at least the major part ofthe precipitate and a minor part of the non-precipitated material, and

f) separating said second fraction comprising the tissue repairpromoting substances from said first fraction within the secondcontainer, and

g) optionally heating said second fraction within one of the containersof the container system so as to dissolve the isolated precipitatedtissue repair promoting substances.

In a further aspect, the invention provides a method for preparing afraction comprising tissue repair promoting substances for a singlehuman or animal individual, said fraction being kept for application toa tissue of the human or animal individual from whom the blood wascollected.

The method may, e.g., be performed as follows:

Blood is collected from a single human or animal individual. The bloodmay be collected from a single healthy donor or from an individual inneed of tissue repair. The blood collection may be performed severalweeks or months prior to use, or, preferably within a short period priorto use.

The blood is collected directly into a collection system comprising atleast two containers interconnected so as to enable transfer of materialfrom one container to another without establishing contact with theenvironment. Transfer of material between the containers of the systemcan be performed by external manipulation of the system, and separationand isolation may be performed by precipitation and centrifugation. Aprecipitation enhancing agent may be added to the system prior to thecollection of the blood.

The method according to the invention permits the preparation of allsubstances within a closed sterilized system. Thus, the resultingfraction for the promotion of tissue repair can be used directly fromone of the containers of the container system without being subjected tolaborious washing or sterilization procedures. The fraction whichcomprises a large amount of fibrinogen is isolated within one of thecontainers and can be used directly as a component in e.g. atissue-adhesive system. The fact that the yield of the fibrinogencomponent is very high provides for the possibility of preparing a largeamount of tissue-adhesive material from only a small amount of blood.

Since the substances obtained are to be used in the treatment of aliving human or animal individual, every step of the procedure must beperformed so as to exclude the possibility of the material beingcontaminated from the environment. One aspect of the present inventionis to provide an apparatus comprising interconnected closed containers,the apparatus permitting the preparation to be performed so that allsubstances are prepared within a closed system. Immediately after thecollection of the biological material, the access to the inner of theapparatus is sealed and during all the processing steps, the system isclosed, i.e. no direct access to the environment is established. Thisfeature of the method and apparatus makes the application possible in aroutine laboratory not having access to facilities usually employedduring operations under sterile conditions. The collection of thebiological material, e.g. the puncturing of a blood vessel, must beperformed by a person skilled in obtaining biological material understerile conditions. Thus, this first step of the procedure may beperformed by e.g. a technician skilled in collecting blood samples frompatients. Immediately after the collection, the collection conduit issealed and during all the subsequent steps the system is sealed from theenvironment, and these steps may therefore be performed by a person notskilled in the sterile techniques.

The advantages of the present invention can be summarized as follows:

the method provides a very high yield of the biologically activesubstances;

the procedure can be performed rapidly, i.e. within approximately 1/2-2hours;

the preparation of the substances may be performed shortly before use,thus problems concerning stability over longer storage periods aretotally avoided;

the whole procedure is performed in such a way that contamination fromthe outside is avoided, i.e. the resulting product is obtained in a formimmediately suitable for therapeutic application;

tissue repair promoting substances from blood cells can be preparedsimultaneously with the preparation of tissue repair promotingsubstances from blood plasma, as explained in greater detail below;

the risk of transmission of microorganisms and immunogens from foreigndonors is minimized.

the tissue repair promoting substances are not subjected to potentiallyactivity reducing procedures such as heating sterilization andlyophilization.

the substances may be prepared at very low costs.

The isolated substances may be used in a tissue-adhesive system, saidsystem comprising as the main component fibrinogen to be used as aprecursor for fibrin which in turn will participate in the formation ofa stable fibrin clot. The composition according to the invention may beapplied simultaneously with a solution containing afibrinogen-activating enzyme such as thrombin, for example applied byway of two syringes connected by tubings adapted so as to ensure thatthe two solutions are not mixed prior to application to the tissue to berepaired. Such an application method provides excellent means foravoiding inconvenient clogging of the application system duringoperation. A further advantage of this application method is that itenables application of the substances at a site distant from thesyringes, i.e. application can be performed for instance to fistulaedistant from the exterior surface of the body or to the wound caused bybiopsying inner organs.

The method permits the simultaneous isolation of tissue repair promotingsubstances derived from blood cells such as platelets, thus conferringvaluable properties to the product. It is well-known that certainfactors such as the platelet-derived growth factors (PDGF) promote theproliferation of mesenchymal cells thus contributing to the process oftissue repair. When the tissue-adhesive system is applied to a tissue towhich the blood perfusion is low and hence the amount of platelets islow, the application of platelet-derived factors may be an advantage.The method and apparatus according to the invention permits thesimultaneous preparation within the same system of several tissue repairpromoting substances, i.e. several substances naturally present in bloodplasma and several substances naturally present in blood cells such asplatelets.

An important advantage of the method according to the invention is thatit is readily applicable in combination with ordinary blood bankroutine. Thus, the method provides the means for preparing tissue repairpromoting substances simultaneously with the preparation of thewell-known blood fractions prepared from donor blood, i.e. at least aminor part of the donated blood material can be used for the preparationof tissue repair promoting substances whereas the remaining bloodmaterial can be used for the preparation of clinically importantfractions such as the erythrocyte containing fraction, the buffy coat(platelets and leucocytes) fraction etc. The container comprising theprepared tissue repair promoting substances may be stored together withthe containers comprising the other prepared blood fractions. Thisprovides for the possibility of administering the blood fractions suchas the erythrocyte fraction and the fraction comprising the tissuerepair promoting substances obtained from one donor to one patient, e.g.a patient undergoing major surgery and thereby needing both atransfusion of erythrocytes and repair of a surgical wound. Thus, therisk of transmitting infectious diseases by administering the tissuerepair substances to an individual in need thereof, is not augmentedcompared to the risk of transmitting infectious diseases via theerythrocyte transfusion which was administered in any case.

The above-mentioned blood bank aspect of the invention providesconsiderable practical and economical advantages:

the blood material is utilized in a way that ensures a minimum loss ofthe valuable blood material;

the routine procedures employed in the blood bank are not disturbed-onthe contrary, the administrative resources being employed in checkingand calling upon the donors, collecting, handling, storing anddistributing the blood material are fully utilized. An important part ofthis is the utilization of the extensive (and expensive) machineryemployed in the testing of the donors for diseases, first of allinfectious diseases such as hepatitis and AIDS;

no extra specialized personnel or equipment is needed for thepreparation,

the friction comprising the tissue repair promoting substances isautomatically classified and marked according to the blood group of thedonor, and this provides for the possibility of administering thecomposition comprising the tissue repair promoting substances inaccordance with this classification.

A further important advantage of the invention is that it permits theisolation of biological substances in such a way that the donor of thestarting material for said substances is identical to the recipient ofsaid isolated substances. Thus, the method makes it possible to isolateblood substances from blood collected from only one donor and furtherapplying the resulting product to a tissue of said donor. By thisprocedure the risk of transferring foreign microorganisms from sickdonors to the recipient, e.g. the risk of transmitting AIDS or hepatitisvirus is totally avoided, and what is also of importance, thetransferred amount of foreign immunogens is minimized.

In another important aspect, the present invention relates to anapparatus for carrying out the method of the invention, comprising firstand second containers of a type defining a reducible inner volume, ablood inlet conduit connected to the first container and including afirst closure valve, a conduit interconnecting the first and secondcontainers and including a second closure valve, and optionally afurther container or several further containers connected thereto and,when several further containers are present, interconnected, by means ofa conduit (conduits) including a closure valve (closure valves).

More specifically, at least one of the containers may be a syringecylinder comprising a piston member displaceably arranged or acollapsible flexible bag. Such a bag may be supplied with meanspermitting the subdivision of the bag into two or more individualchambers, in particular pinching means.

DETAILED DESCRIPTION OF THE INVENTION

The collection of blood may be performed within a few hours such as 1/2or 1 hour, or the day before the use of the tissue repair promotingsubstances. Alternatively, the preparation may be performed severalweeks before use of the preparation.

The blood is collected from a vein of the donor, preferably bypuncturing the vein by means of a sterilized hollow needle, said needlebeing connected to a tubing which in turn is connected to the firstcontainer of the collecting system according to the invention.Alternatively, the blood may be collected from an outlet device mountedto the one end of a catheter placed in the vein of a donor--thisalternative being applicable if sterile conditions are ensured duringthe collection.

The amount of collected material can be adapted to suit the particularpurpose. Thus, if the blood is to be collected from e.g. a healthyvoluntary donor the usual amount of collected blood is in the order of400-600 ml, and at least part of said amount may be used for thepreparation of tissue repair promoting substances.

If the blood is to be collected from a donor in need of treatment withthe tissue repair promoting substances, the volume of the collectedblood may appropriately constitute no more than a few % of the donor'stotal blood volume. If the subject to be treated is an adult humanbeing, for instance 25-50 ml blood will be appropriate for theproduction of a preparation suited for most purposes.

The components of the container system is preferably made from materialwhich optimally does not exude damaging substances to the biologicalmaterial contained within the system. Furthermore, the material shouldbe able to tolerate sterilization procedures usually employed formaterial which is to be used for clinical purposes. The sterilizedsystem may, apart from the pathway used to collect the biologicalmaterial, be constructed so as to exclude during operation directcontact between the internal spaces of the closed system and theenvironment.

Depending on the method selected for the fractionation of the bloodobtained, the container system may comprise from two to six or moreinterconnected containers. Any agents needed during the preparation ofthe various fractions may be supplied to the containers of the containersystem prior to collecting the blood. The container system may then besterilized after supplying the containers with the various agents so asto maintain the sterility of the spaces defined by the system.

The blood is collected into a first container of a container systemcomprising first and second interconnected containers, in some casesmore interconnected containers. Since the first container of thecontainer system is to be used for the blood collection, it shouldcontain anticoagulants. Some of the conventional anticoagulants may beemployed: citrate, acid-citrate dextrose (ACD),citrate-phosphate-dextrose (CPD), or ethylene diamine tetra-acetic acid(EDTA). Furthermore, heparin may be added in an amount sufficient forthe prevention of thrombin activity during the processing steps.Proteolytic enzyme inhibitors, such as aprotinin e-aminocaproic acid ortranexamic acid may be added in order to prevent proteolytic degradationof the substances.

The container used for collecting blood may be collapsible flexible bag,preferably a plastic bag. Alternatively the first container may be asyringe cylinder comprising a displaceably arranged piston member.

During collection of the blood into the first container, said containeris preferably gently agitated to mix the blood with the chemical agentssupplied to said first container of the system. After finishing theblood collection the tubing previously used for the blood collection issealed. The sealing may be obtained by pinching the tubing with e.g. aclip. Alternatively, a closure valve may be positioned so as to blockthe contact between the inner space of the container system and theenvironment. The container system may e.g. comprise closure valves to beused in directing the liquid flow in the containers and tubings of thesystem. The tubing employed in the collection of the blood may at thisstage be disconnected from the container system.

The container system is subsequently centrifuged in a conventional wayso as to separate the blood in various fractions. The container systemmay be placed in a centrifuge holder without any of the interconnectedcontainers being disconnected. This may be obtained by employing aspecially adapted centrifuge holder. By adjusting the gravity forcesemployed, the blood may be separated into the following fractions:

a first fraction essentially comprising plasma and a second fractionessentially comprising blood cells including the major part of theerythrocytes, or

a first fraction essentially comprising plasma and a minor fraction ofsome of the blood cells such as the platelets, and a second fractioncomprising the major part of the blood cells including the major part ofthe erythrocytes, or

a first fraction essentially comprising plasma, and a second fractioncomprising essentially a part of the blood cells such as the plateletsand leucocytes, and a third fraction comprising at least the major partof the erythrocytes.

After the centrifugation, parts of the fractions obtained by thecentrifugation are transferred to the other containers of the containersystem and the tissue repair promoting substances are isolated withinthe containers.

The important step for isolating tissue repair promoting substancesnaturally present in the plasma of normal human or animal blood is astep comprising precipitation of said substances. These substances maybe fibrinogen, factor VIII, factor XIII, and fibronectin. Theprecipitation may be obtained by subjecting the plasma-containingfraction to a low temperature, e.g. temperatures below 10° C.,preferably below 4° C., more preferably in the interval from -80° C. to2° C., especially in the interval from -30° C. to -10° C. Wheneverfrozen fractions are obtained, a step comprising thawing is employed,e.g. thawing the fractions at temperatures in the interval from -3° C.to 4° C.

The precipitation may be promoted by adding a precipitation promotingagent to the plasma fraction prior to or during subjection of the plasmafraction to a low temperature. The agent may be a lower alkanol such asa C₁₋₄ -alkanol, e.g. methanol, ethanol or isopropanol, preferablyethanol, e.g. in a concentration of 1-30% w/w such as 3-20% w/w,preferably 6-11% w/w; or a lower alkyl ether such as diethyl ether in aconcentration of 5-12% w/w, preferably 10-11%. Additionally, the agentmay be an amino acid, e.g. lysine or glycine in a concentration of0.1-3M, preferably 2.2M. The pH in the solution during the precipitationis preferably in the range of 5-9, preferably in the range of 6-8. Theprecipitation may also be promoted by changing the ionic strength of thesolution by way of adding various salts such as ammonium sulphate or byadding polyethylene glycol or ethacridine. The precipitation promotingagents described above may be used alone or in combinations comprisingtwo or more of the various agents. When suitable chemical agents areadded, the precipitates may optionally be obtained without freezing thefractions, e.g. employing temperatures in the interval from -3° C. to10° C., preferably from -3° C. to 4° C.

The tissue repair promoting substances naturally present in blood cellsmay be obtained from the cells by subjecting the cells to variations intemperature. A procedure involving freezing and subsequent thawing ofthe cells causes disruption of the cellular structures thereby releasingthe comprised substances. Thus, growth factors may be released fromplatelets by subjecting the platelets to freezing and thawingprocedures. Furthermore, specific agents added to the cell-containingplasma fractions may cause release of certain tissue repair promotingsubstances. The addition of substances such as adenosine diphospate,adrenalin, thrombin or collagen to platelets may cause the release ofplatelet-derived growth factors. Thus, the word "release" should, in thepresent description and the accompanying claims, be understood to applyto both specific cellular release and to the release of substancesobtained by disruption of the cells, obtaining e.g. a cellular extract.

A way to concentrate the precipitate within the second container is touse natural gravity forces to sediment the precipitate within saidsecond container, i.e. allowing the container to stand for a while inorder to sediment the precipitate. Alternatively, the precipitate isconcentrated in the second container by means of centrifugation wherebythe precipitate is concentrated in the pellet obtained and thusseparated from the non-precipitated material comprised in thesupernatant.

The second fraction comprising the precipitated tissue repair promotingsubstances may be isolated within the second container by dischargingthe first fraction comprising the major part of the non-precipitatedmaterial. Said discharging may be carried out without establishing anycontact to the environment by transferring the first fraction to a thirdcontainer of the container system.

In another preferred method according to the invention, the secondfraction is isolated from the first fraction within the second containerby dividing the volume of the second container into first and secondchambers containing said first and second fractions, respectively. Theisolation of the second fraction within the second container may becarried out by employing a container comprising collapsible flexiblewall parts as the second container of the container system and dividingthe volume of said second container into first and second chambers bypinching said flexible wall parts.

The second or third container of the container system used in the methoddescribed above, may preferably be supplied with precipitation agentsprior to use of said system.

Another preferred method according to the invention is to prepare afraction comprising tissue repair promoting substances obtained fromboth the blood plasma and the blood cells. This is obtained by employingthe following sequential steps:

a) centrifuging the container system containing the blood so as toseparate the blood into various fractions including a cell-containingplasma fraction,

b) transferring at least part of said cell-containing fraction to thesecond container of the container system,

c) sedimenting the cells at the transferred fraction within the secondcontainer,

d) making a primary temporary division of the volume of the secondcontainer, said division dividing the second container into a firstchamber containing the sedimented cells and a minor part of the plasmaand a second chamber containing the major part of the plasma, saiddivision preferably being obtained by pinching the wall parts of thesecond container,

e) subjecting the second container to a low temperature so as to obtaina precipitate in the second chamber, and so as to disrupt the cellularstructures of the cells contained in the first chamber thereby releasingtissue repair promoting substances from the cells, thus obtaining afraction enriched with tissue repair promoting substances released fromthe cells,

f) sedimenting the precipitate within the second chamber, thus obtaininga second fraction within said second chamber, said second fractioncomprising at least the major part of the precipitate and a minor partof the non-precipitated material, and at the same time sediment themajor part of the cells and the cell residues within the first chamber,

g) further subdividing said first chamber into a first "waste chamber"containing the sedimented cell residues and a third chamber containing athird fraction, said third fraction comprising at least the major partof the fraction enriched with tissue repair promoting substancesreleased from the cells and being substantially free of cells and cellresidues,

h) further subdividing the second chamber into a second "waste chamber"containing the major part of the non-precipitated material and a fourthchamber containing at least the major part of the second fraction, and

i) optionally, abolishing the primary temporary division of the secondcontainer, e.g. by removing the pinching of the wall parts, thusobtaining a combined chamber comprising the former third chamber andformer fourth chamber. In doing so, the third fraction enriched withtissue repair promoting substances released from the cells was combinedwith the second fraction comprising the major part of the precipitatewithin the same container,

j) and optionally heating the second fraction or the optionally combinedfraction within the second chamber or within the optionally formedcombined chamber so as to dissolve the precipitated tissue repairpromoting substances.

In a preferred embodiment of the invention, the second container of thecontainer system includes flexible wall parts, and the divisions of thevolume of the second container are performed by pinching said flexiblewall parts.

Thus, minimum loss of material during the procedures was ensured, thisspecial feature of the method adding to the excellent high yield ofbiologically active substances.

In the method of the invention, precipitation promoting agents can beadded to the plasma fraction contained in the second chamber prior to orduring subjecting the second chamber to a low temperature. Said agentsmay be contained in a third container of the container system until theprimary temporary division of the second container has been carried out.

Furthermore, specific agents suited for promoting the release of tissuerepair promoting substances from blood cells may be added to thefractions comprising the cells. These agents may be supplied to acontainer of the container system prior to use. Said specific agents maybe employed in combination with the obtainment of the cellular extractsby means of freezing/thawing procedures.

In a preferred method, a fraction comprising a major part of the bloodplasma and a major part of the platelets is transferred to the secondcontainer, and subsequently tissue repair promoting substances derivedfrom both the blood plasma and the platelets are isolated within thechambers of the second container, and optionally finally combined.

If, for some reason, the second and third fractions obtained in thechambers are needed separately, the combination step is omitted.Furthermore, the method and container system according to the inventionmay be used solely for preparing tissue repair promoting substancesderived from blood cells, i.e. the plasma precipitation steps may beomitted, and the plasma may be used for alternative purposes ordiscarded.

Another preferred method comprises the following steps:

a) collecting the blood into a first container of a container systemcomprising at least first, second, third, and fourth containers,

b) centrifuging the container system containing said blood so as toseparate the blood in various fractions including a substantiallycell-free plasma fraction and a cell-containing plasma fraction,

c) transferring at least part of the substantially cell-free plasmafraction to the second container of the container system,

d) transferring at least part of the cell-containing plasma fraction tothe third container of the container system,

e) subjecting the substantially cell-free plasma fraction in the secondcontainer to a low temperature so as to obtain a precipitate comprisingtissue repair promoting substances derived from plasma,

f) concentrating said precipitate within the second container so as toobtain a first fraction comprising a major part of the non-precipitatedmaterial and a second fraction comprising at least the major part of theprecipitate and a minor part of the non-precipitated material, andseparating said second fraction comprising the tissue repair promotingsubstances from the first fraction within the second container,

g) optionally discharging the first fraction to the fourth container,

h) subjecting the cell-containing fraction in the third container to aprocedure which promotes the release of tissue repair promotingsubstances from the cells thus obtaining a fraction enriched with tissuerepair promoting substances from the cells,

i) sedimenting the cells and cell residues within the third containerthus obtaining a third fraction enriched with tissue repair promotingsubstances and substantially free of cells and cell residues,

j) separating said third fraction from at least the major part of thesedimented cells and cell residues within the third container andoptionally transferring at least part of said third fraction to thesecond container, thus combining the third and second fraction, and

k) optionally heating the optionally combined fraction within the secondcontainer so as to dissolve the precipitated tissue repair promotingsubstances.

In a preferred embodiment of the invention, the third fraction isseparated from at least the major part of the sedimented cells and cellresidues by dividing the volume of the third container into first andsecond chambers containing said third fraction and the major part of thesedimented cells and cell residues, respectively. The third containermay include flexible wall parts and the third container may be dividedby pinching said flexible wall parts.

The sedimentation rate may be increased by means of centrifugation.

The procedure for promoting the release of tissue repair promoting,substances may be a procedure involving freezing and thawing of thecells so as to disrupt the cellular structures of the cells and therebyreleasing the tissue repair promoting substances. Furthermore, chemicalagents suitable for promoting release of tissue repair promotingsubstances may be added to the cell-containing fraction.

The method may be carried out so that the cell-containing plasmafraction is a platelet-containing plasma fraction, e.g. comprising themajor part of the platelets.

An important aspect of the method is that a sterilized container systemmay be employed, and the collection of the blood in the first containerand the further steps identified are performed without establishing anydirect contact between the fractions within the container system and theenvironment so as to obtain the fractions comprising the tissue repairpromoting substances free from any contamination from the environment.

The precipitation promoting agents may be added to the plasma fractionprior to or during the subjection of the second container to a lowtemperature.

The tissue repair promoting substances which may be prepared from theplasma fraction are fibrinogen, factor VIII, factor XIII, andfibronectin.

The tissue repair promoting substances released from platelets maycomprise platelet-derived growth factor and epidermal growth factor.

Optionally, these fractions may be used separately or a combinedfraction may be isolated within one of the containers of the containersystem, said combined fraction comprising e.g. fibrinogen, factor VIII,factor XIII, fibronectin, and substances derived from platelets.

Furthermore, a tissue repair promoting composition comprisingfibrinogen, factor VIII, factor XIII, fibronectin and substances derivedfrom platelets, the composition being substantially free of fibrin, maybe prepared according to the method of the invention.

The compositions according to the invention may be used for thepromotion of tissue repair in a human or animal individual.

A further aspect of the invention is to provide a method for isolatingtissue repair promoting substances including fibrinogen from human andanimal blood, which method comprises

a) collecting a blood sample from the human or animal individual,

b) recovering more than 60% of the fibrinogen present in the bloodsample in a form suited for application to a human or animal individual,the fibrinogen typically being concentrated approximately 10-20 times.

The fibrinogen concentration of the compositions prepared according tothe methods of the invention usually is in the range of 20-45 mg/ml,often 25-35 mg/ml. Thus, the fibrinogen is concentrated approximatelyfrom 10 to approximately 20 times the concentration in plasma. Solutionswith fibrinogen concentrations exceeding approximately 40 mg/ml may havea tendency to gelate at room temperature. However, this problem iseasily solved, since resolution is achieved by incubating the solutionat e.g. 37° C. for a few minutes.

The yield of fibrinogen is usually in the range of 60-95%, often morethan 70%, usually approximately 70-85%, of the fibrinogen content in thestarting material, the fibrinogen being quantified by means of afunctional assay measuring the amount of biologically active fibrinogen.

The concentration of factor XIII in the solution prepared was usually inthe order of 20-25 times the concentration of factor XIII in the plasmafraction of the blood samples collected.

A further aspect of the invention is to provide a method for promotingthe repair of a tissue of a human or animal individual, which methodcomprises

a) collecting a blood sample from the human or animal individual,

b) preparing tissue repair promoting substances from said blood sample,and

c) applying said prepared tissue repair promoting substances to thetissue of said human or animal individual.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described with reference to thedrawings, in which

FIG. 1 is a diagrammatic side view of a first, presently preferredembodiment of an apparatus according to the invention.

FIG. 2 is a diagrammatic side view of a second embodiment of anapparatus according to the invention,

FIG. 3 is a sectional view of a modified embodiment of a container ofthe apparatus shown in FIG. 1 or 2,

FIG. 4 is a diagrammatic side view of the two syringes used to apply thetwo substances used for tissue repair.

DETAILED DESCRIPTION OF THE DRAWINGS

In FIG. 1 a preferred embodiment of an apparatus or system according tothe invention is shown designated 10 in its entirety. The isolatingdevice 10 comprises two or more containers connected by means of tubingsand valves for directing and stopping the flow of the liquids containedin the apparatus. The apparatus is preferably made from suitablematerials such as plastics materials, which are preferably capable ofwithstanding sterilizing procedures or processes such as radiationtreatment including UV radiation, or hydrogen peroxide treatment.

In FIG. 1 the system 10 comprises 4 containers which in their entiretyare designated 11, 23, 31, and 37. The containers 11, 23, 31 aresyringes, i.e. containers suitable for manipulating the content by meansof operating a piston, whereas the container 37 is a bag of collapsibleflexible plastics material. Alternatively two or one of the containersmay be syringes comprising cylinders and pistons whereas two or threemay be bags, such as collapsible flexible plastic bags. The bag or bagsmay be provided with outlet/inlet devices suitable for mountingsterilized tubings in a way which ensures that no contamination from theenvironment occurs. All the containers of the container system may bemade of collapsible flexible plastic bags, preferred embodiments of theinvention comprising four or five such bags. Furthermore, the containersystem may comprise five or more interconnected containers, some or allof the containers being bags, or some or all of the containers beingsyringes.

The pistons of the syringes may each be provided with screw couplingmeans to permit disconnection of the piston rod when the syringe isfilled and is to be centrifugated. Alternatively, the syringes may beprovided with piston rods being so short that when the piston rod isretracted to the desired position, the base of the piston flushes thebase of the syringe cylinder so as to avoid further retraction duringcentrifugation. Devices for supporting the piston during centrifugation,especially when some of the syringe cylinders are empty or partlyfilled, may be employed. Thus, the piston rod may be used to manipulatethe piston during emptying/filling of the syringe cylinder and furtherto support the piston during centrifugation. Another alternative is toemploy a syringe comprising a piston provided with a string (instead ofa piston rod) to be used in the manipulation of the piston.

In FIG. 1, the first syringe 11 comprises a syringe cylinder 12, apiston 13 and a piston rod 14, the piston 13 and piston rod 14 beingprovided with cooperating screw coupling means, bayonet coupling meansor the like to permit disconnection of the piston rod 14 from the pistonduring centrifugation of the system. The piston 13 and the syringecylinder 12 define a collecting chamber 15 suitable for storage of thecollected biological material. The volume defined by the chamber 15 may,for example, be approximately 50 ml when the piston is maximallyretracted from the delivery end 16, but it is, of course, also possibleto have the volume smaller or larger, e.g. in the range from about 10 mlto about 200 ml or even 500 ml.

The delivery end 16 of the syringe 11 is connected to a three-way valve17 which is connected to a tubing 18. The tubing 18 is provided with ahollow needle 19 suitable for puncturing a reservoir containingbiological material, e.g., a blood vessel.

The three-way valve 17 is further connected to a tubing 20. The tubing20 is connected to a three-way valve 21 which is connected to a deliveryend 22 of a second syringe 23. The syringe 23 comprises a syringecylinder 24, a piston 25 and a piston rod 26. The piston 25 and thecylinder syringe 24 define a chamber 27. The volume defined by thechamber 27 may, for example, be approximately 5 ml when the piston ismaximally retracted from the delivery end 22 of the syringe 23, butanalogously to what is stated above concerning the volume of the chamber15, the volume of the chamber 27 may be smaller or larger, for examplein the range from about 1/2 and 1 ml to about 20 ml or 50 ml, the volumeof the chamber 27 normally being adapted to the volume of the remainingsystem, such as the volume of the chamber 15.

The three-way valve 21 is further connected to a tubing 28. The tubing28 is connected to a three-way valve 29 which is further connected to adelivery end 30 of the syringe 31. The syringe 31 comprises a syringecylinder 32, a piston 33 and a piston rod 34, which may be constructedin correspondence to the syringe cylinder 12, the piston 13, and thepiston rod 14, respectively, of the first syringe 11. The piston 33 andthe inner of the syringe cylinder 32 define a chamber 35. The volumedefined by the chamber 35 may, for example, be approximately 30 ml whenthe piston is maximally retracted from the delivery end 30, butanalogously to what is stated above, the volume may be smaller orlarger, e.g. in the range from about 6 ml to about 120 ml or even 300ml, the volume being adapted to the dimensions of the other containersof the system. The three-way valve 29 is further connected to a tubing36 which is connected to a bag 37.

The volume comprised by the bag may be approximately 40 ml but, asstated above, adapted to the dimensions of the other containers of thesystem.

In FIG. 2 a second embodiment of an apparatus or a system according tothe invention is shown. The second embodiment of the apparatus differsfrom the above-described, first embodiment of the apparatus according tothe invention in that the three-way valves 17, 21 and 29 are omitted.The tubings 18 and 20 are consequently connected directly to the firstsyringe 11, and the tubings 20 and 28 are also connected directly to acontainer 71. The container 71 is a flexible and collapsible bag of aplastics material such as a polyvinyl chloride bag. In the secondembodiment of the apparatus or system according to the invention, thefirst syringe 11 comprises a string 72 which is connected to the pistonrod 14 of the syringe. The piston rod 14 shown in FIG. 2 is somewhatshorter than the piston rod 14 shown in FIG. 1. - 73, 74 and 75 showsites for pinching the tubings so as to block the liquid flow throughthe tubings. 76 shows a site for pinching the container 71.

FIG. 3 shows an embodiment of a syringe to be employed in the apparatusor system shown in FIG. 1 or FIG. 2. The syringe is designated 50 in itsentirety and comprises a syringe cylinder 51, a piston 52 and a pistonrod 53. The piston 52 and the syringe cylinder 51 define a chamber 54. Aflexible plastic lining 55 is attached to the piston 52 and the open endof the syringe cylinder 51. During manipulation of the piston 52, thepiston end of lining 55 moves with the piston 52, thus preventingcontact between the collecting chamber 54 of the syringe cylinder 51 andthe environment.

In FIG. 4 an apparatus or system, designated 60 in its entirety forapplication of a composition comprising tissue repair promotingsubstances is shown. The apparatus is used for the application of twocomponents of a tissue-adhesive system comprising as a first component afibrinogen-containing solution and as a second component anenzyme-containing solution.

The system comprises a first syringe 61 suitable for storing one of thesubstances of the two-component tissue-adhesive system and a secondsyringe 62 for storing the other component of the tissue-adhesivesystem. A needle 63 is connected to a delivery end of the first syringe61, and a needle 64 is connected to a delivery end of the second syringe62. A tubing 65 has its first end connected to the needle 63 and itssecond end connected to the needle 64. The tubing 65 is further providedwith an intermediate puncture 70. The puncture 70 serves the purpose ofrendering it possible to expel the two components contained in thesyringes 61 and 62 at an application site of a human or animalindividual. The first syringe 61 comprises a piston rod 67, and thesecond syringe 62 comprises the piston rod 68. The syringes 61 and 62are supported through a supporting device 66, and the piston rods 67 and68 are interconnected through a connection device 69 so as to render itpossible to operate the piston rods 67 and 68 simultaneously.

THE APPLICATION OF THE TISSUE REPAIR PROMOTING SUBSTANCES

The apparatus shown in FIG. 4 was used for the application of the twocomponents of a tissue-adhesive system.

A syringe 61, the total volume of which was approximately 1 ml and asyringe 62, the total volume of which was approximately 3 ml, were used.The syringe 62 contained the solution comprising the tissue repairpromoting substances according to the invention. The syringe 61comprised a solution containing the enzyme thrombin (e.g. obtained fromHoffmann La-Roche) in a concentration of 1200 NIH-U/ml, and furthercomprising 100 mM Ca⁺⁺ and 7000 KIE/ml aprotinin (Trasylol®, Bayer). Theconcentration of thrombin could be reduced to 5-10 NIH-U/ml when a slowconversion of fibrinogen was desired. By application of thefirst-mentioned solution, fibrinogen could be converted to fibrin within5-15 sec.

The two solutions could be expelled from the system by simultaneouslypressing the piston rods 67 and 68.

When the system is to be used to apply solutions at an application sitein the interior of the human or animal individual, the tubing 65 may beas long as e.g. more than 1 m, the syringes (or other pressure deliverysystems) being adapted thereto. Such a system may be applicable in casesin which tissue adhesion is needed e.g. subsequent to surgical biopsy inthe interior of the human or animal body. In this case, the tubing 65may be placed in a catheter and transferred within this catheter to theapplication site.

If the fibrinogen containing solution is gelatinating due to highconcentration of fibrinogen, fluidity may be obtained within a fewminutes by immersing the whole system into a sterilized water bath atapproximately 37°-40° C.

By means of the application apparatus, the two solutions may be keptseparate until they are expelled from the system, i.e. no coagulationwill take place inside the tubings or needles. (When the applicationsystem is not in use, a clot may be formed at the puncture 70 of thetubing 65, but this may easily be removed).

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1

Preparation of a composition containing tissue repair promotingsubstances from blood from an adult human donor using a sterilizedcontainer system comprising four containers

For the preparation, a system corresponding to the system shown in FIG.1 was used. Tissue repair promoting substances present in blood plasmaand cells, first of all platelets, were isolated.

Sterile chemical agents which were to be used in the preparation werefilled into sterilized syringes in order to obtain a sterile system.

Syringe 11 contained 6 ml 113 mM tri-sodium citrate solution and 20IE/ml heparin (Leo) as anticoagulants and 800 KIE/ml aprotinin as enzymeinhibitor.

Syringe 23 contained 5×10⁻⁶ mol freeze-dried adenosine 5-diphosphate(ADP) to be used in promoting growth factor release from platelets.

Syringe 31 contained 2.5 ml 93% (w/w) ethanol to be used as a chemicalagent suitable for obtaining a precipitate comprising tissue repairpromoting substances.

44 ml blood was collected from a human adult donor. The hematocrit valuewas measured to be 0.46, i.e. said blood diluted with the anticoagulantcomprised 31 ml plasma fraction.

The puncture of the vein was performed by means of the needle 19, andthe blood was transferred to the syringe 11 via the tubing 18. Duringthe collection of blood and the subsequent filling of the syringe 11,the syringe was gently agitated to mix the blood with the anticoagulant.

After collection of the blood, the system was disconnected from thedonor and the tubing 18 was sealed. The system was placed in anespecially adapted centrifuge holder, said holder comprisingcompartments defining volumes suitable for the various containers of thesystem. Thus, the complete container system, including the tubings,could be placed in the centrifuge holder. The syringes were placed sothat the delivery ends pointed upwards during centrifugation. The systemwas centrifuged for ten minutes at 2000×g at 15° C. in order to obtainwithin the syringe 11 a substantially cell-free plasma fraction, acell-containing fraction (comprising platelets and luecocytes and aminor part of the erythrocytes), and a cell-containing plasma fractioncomprising the major part of the erythrocytes.

The volume of the substantially cell-free plasma fraction wasapproximately 25 ml. By gently pressing the piston 13 towards thedelivery end 16, 22 ml of substantially cell-free plasma fraction weretransferred to the syringe 31. During this transfer the closure valves17, 21 and 29 were positioned so as to direct the liquid flow into thesyringe 31.

Subsequent to the transfer of the substantially cell-free plasmafraction, the closure valves 29 and 21 were positioned so as to directthe liquid flow into the syringe 23. The cells of the cell-containingplasma fraction comprising platelets, leucocytes and a minor part of theerythrocytes were stirred up in the remaining 3 ml plasma. By pressingthe piston 13 towards the delivery end 16, said cell-containing plasmafraction was transferred to the ADP-containing syringe 23. The syringe11 was disconnected and the biological material was either discarded orused for alternative purposes such as transfusion of the erythrocytefraction to the human donor from whom the blood had initially beentaken.

The syringes 23 and 31 were treated so as to obtain thorough mixing ofthe materials contained therein. During this period, the ADP promotedthe release of tissue repair promoting substances such as PDGF. Thewhole system was subsequently incubated in an ice bath at 0° C. forapproximately 30 minutes. During this period the ethanol precipitationtook place in the syringe 31. If optionally the syringe 23 is incubatedin e.g. an ice bath, the low temperature causes a partial disrupture ofcellular structures of the cells, first of all the platelets, present inthe syringe 23, thereby releasing tissue repair promoting substancesfrom the cells, including e.g. the platelet-derived growth factor andthe epidermal growth factor.

After the incubation period, the container system was centrifuged for 10minutes at 2000×g at approximately 0° C. Thus, the precipitate obtainedin the syringe 31 was comprised in a firm pellet separated from theremaining part of the plasma fraction. At the same time, the cells andcell residues within the syringe 23 were sedimented, and thus a fractionenriched with tissue repair promoting substances from the cells,primarily platelets, (a "cell extract") was obtained.

The syringe 31 was turned upside down, i.e. the delivery end 30 pointeddownwards. The closure valve 29 was positioned so as to direct theliquid flow from the syringe 31 to the tubing 36 of the container 37. Bypressing the piston 33 towards the delivery end 30, the non-precipitatedplasma material was transferred to the container 37 via the tubing 36.During this procedure, the air present in the syringe 31 facilitated theseparation of the plasma fraction from the sedimented precipitate.

The closure valves 21 and 29 were positioned so as to direct the liquidflow from the syringe 23 to the syringe 31. By pressing the piston 25towards the delivery end 22 of the syringe 23, a part of the cellextract, typically approximately 0.2 ml, sufficient for obtaining adissolved precipitate, was transferred to the tubing 28 and subsequentlyinto the collecting chamber 35 of the syringe 31. Thus, the tissuerepair promoting substances to be used were contained in the chamber 35of the syringe 31. The syringe 31 was incubated at 37° C. in order todissolve the precipitate in the cell extract. If needed, further amountsof cell extract could be added in order to facilitate dissolution of theprecipitate.

After approximately 10 minutes the precipitate was dissolved, andapproximately 1.5 ml of the solution comprising the tissue repairpromoting substances was obtained. This solution could be usedimmediately or alternatively stored until use, e.g. stored in a frozenstate.

The resulting solution contained 26 g/l of fibrinogen, and the recoveryof fibrinogen was 84% of the amount present in the 22 ml plasma fraction(containing 2.1 g/l), or 60% of the amount present in the 44 ml of bloodcollected. The method used for determination of fibrinogen was based onthe conversion of fibrinogen into fibrin which was subsequently isolatedand assayed. (cf. O. D. Ratnoff et al., J. Lab. Clin. Med. 37, 1951, p316; W. D. Sawyer et al., Thromb. Diath. Haem. 5, 1961, p 149; and T.Astrup et al., Scand. J. Clin. & Lab. Invest. 17, 1965, 57). The assayis a "functional assay" in that only the fibrinogen which is convertibleto fibrin is determined. Thus, the values representing the fibrinogenrecoveries indicate the recovery of biologically active fibrinogen, i.e.fibrinogen useful in obtaining tissue adhesion.

The platelets comprised in the 3 ml plasma were concentrated from the 44ml of whole blood. 0.2 ml of the cellular extract contained growthfactors corresponding to maximally 2.9 ml of whole blood.

EXAMPLE 2

Preparation of composition containing tissue repair promoting substancesfrom blood from an adult human donor using a sterilized container systemcomprising three containers.

For the preparation a sterilized container system corresponding to thesystem shown in FIG. 2 was used. Tissue repair promoting substancespresent in blood plasma and cells (first of all platelets) wereisolated.

The syringe 11 contained various chemical agents (cf. Example 1), thecollapsible bag 71 was empty, and the syringe 31 contained ethanol (cf.Example 1).

Blood was collected from a human adult donor in a way similar to the waydescribed in Example 1.

After collection of the blood, the system was disconnected from thedonor and the tubing 18 was sealed by pinching the tubing 18 with analuminium clip at the site 73. The container system was placed in acentrifuge holder without any of the containers being disconnected fromeach other. The system was centrifuged for 15 minutes at 250×g at 15° C.in order to obtain a platelet-enriched plasma fraction. Thus, the majorpart of the supernatant was constituted by this fraction, and the majorpart of the pellet was constituted by the erythrocytes.

By gently pressing the piston rod 14 towards the delivery end 16, themajor part of the platelet-enriched plasma fraction was transferred tothe container bag 71. During this transfer, the passage through thetubing 28 was blocked by an aluminium clip pinching the tubing 28 at thesite 75.

Subsequent to the transfer of the platelet-enriched plasma fraction, analuminium clip was used for pinching the tubing 20 at the site 74. Thesyringe 11 was disconnected and the biological material was eitherdiscarded or used for alternative purposes such as transfusion of theerythrocyte fraction to the human donor who had contributed the blood.

The container system was centrifuged for 10 minutes at 2000×g at 15° C.in order to sediment cells, first of all platelets, within the secondcontainer 71.

The volume of the container bag 71 was temporarily divided into a firstchamber containing the sedimented platelets and a minor part of theplasma and a second chamber-containing the major part of the plasma.This division was obtained by pinching the walls of the container 71.Subsequently, the aluminium clip at the site 75 of the tubing 28 wasremoved while the ethanol content of the syringe 31 was transferred tothe second chamber of the container bag 71 by pressing the piston rod 34towards the delivery end 30 of the syringe 31. After the transfer, thealuminium clip was replaced at the site 75 of the tubing 28, and thesyringe 31 could subsequently be disconnected. The platelets wereresuspended in the plasma present in the first chamber of the containerbag 71 by shaking the container 71.

The whole system was incubated at -20° C. so as to precipitate thetissue repair promoting substances in the second chamber, and so as todisrupt the cellular structures of the cells contained in the firstchamber thereby releasing tissue repair promoting substances from theplatelets, thus obtaining a fraction enriched with tissue repairpromoting substances released from the platelets. Subsequently, thesystem was subjected to approximately 2° C. for approximately 20 minutesand centrifuged at 2000×g at approximately 0° C. for 15 minutes in orderto sediment the precipitate within the second chamber and the cells andcellular residues within the first chamber.

The chambers were further subdivided by pinching, e.g. by means ofaluminium clips, the flexible walls of the container 71, first bypinching just above the precipitate and then pinching the wall partsjust above the pellet constituted by the cells and cellular residuesadhering to the bottom part of the bag 71. The primary temporarydivision of the second container was abolished, obtaining a combinedchamber comprising the third fraction enriched with tissue repairpromoting substances released from the platelets and the second fractioncomprising the major part of the precipitate. Thus, all the desiredtissue repair promoting substances were now contained in the combinedchamber formed between the two pinching sites. The container 71comprising the combined chamber was incubated at 37° C. in order todissolve the precipitate in the platelet extract. (Alternatively, thecontainer 71 could be stored until use prior to this incubation step.)For further details concerning dissolving time and storage time, videExample 1.

EXAMPLE 3

Preparation of a composition containing tissue repair promotingsubstances from blood from an adult human donor using a sterilizedcontainer system comprising four containers.

For the preparation a sterilized system corresponding to the systemshown in FIG. 1 is used, with the exception that all four containers arecollapsible flexible plastic bags comprising volumes adapted to thefractions obtained, said bags being interconnected by tubings comprisingvalves. Tissue repair promoting substances naturally present in plasmaand in platelets are isolated.

Chemical agents which are to be used in the preparation are filled intothe containers, and the whole system comprising the agents is sterilizedprior to use.

The syringe 11 contains agents as described in Example 1.

The syringe 23 and 37 are empty.

The syringe 31 contains ethanol as described in Example 1.

Human blood is collected from a voluntary donor as described in Example1.

The system is centrifuged so as to obtain a fraction essentiallycomprising erythrocytes, a fraction essentially comprising theleucocytes, and a fraction comprising a platelet-enriched plasmafraction.

The platelet-enriched plasma fraction is transferred to the container37.

The fraction essentially comprising leucocytes is transferred to thesyringe 23.

The syringe 11 essentially comprising the erythrocytes and the syringe23 essentially comprising the leucocytes are disconnected and thebiological materials may be used for alternative purposes.

The system comprising the syringe 31 and the container 37 is centrifugedso as to sediment the platelets in the container 37. The volume of thecontainer 37 is divided so as to obtain a first chamber comprising theplatelets and a minor part of the plasma and a second chamber comprisingthe major part of the plasma. The ethanol comprised in a syringe 31 istransferred to the second chamber of the container 37. The compositioncomprising tissue repair promoting substances derived from plasma andplatelets is isolated within the container 37 as described in Example 2.

As a further modification of the method described in this Example, thecontainer system may comprise a fifth container containing SAG-Msolution (e.g. per 100 ml: sodium chloride 877 mg, anhydrous dextrose818 mg, mannitol 525 mg, adenin 16.9 mg). The SAG-M solution may betransferred to the residual erythrocyte fraction (i.e. the erythrocytefraction obtained in the syringe 11 after the removal of theplatelet-enriched fraction and the leucocyte and platelet comprisingfraction). The addition of the SAG-M solution to the erythrocytefraction is useful when this fraction is to be preserved fortransfusion.

EXAMPLE 4

Preparation of a composition containing tissue repair promotingsubstances from blood from a human donor using a sterilized containersystem comprising two containers.

For the preparation a sterilized system corresponding to the systemshown in FIG. 2 was used, with the exception that the syringe 31 isomitted. The syringe 11 is a syringe and the container 71 is a flexibleplastic bag. Tissue repair promoting substances naturally present inplasma are isolated.

The syringe 11 contains agents as described in Example 1.

The container 71 contains 2.5 ml 93% (w/w) ethanol.

The human blood is collected as described in Example 1. The system iscentrifuged in order to obtain a first fraction essentially comprisingplasma and a second fraction essentially comprising the blood cells.

The plasma fraction is transferred to the container 71, the transferbeing similar to the transfer described in Example 2. The tubing 20 issealed by pinching with an aluminium clip at the site 74. The syringe 11may then be disconnected, and the material used for alternativepurposes.

The container 71 is incubated in an ice bath at 0° C. for approximately30 minutes. During this period, the ethanol precipitation takes place inthe container 71.

After the incubation period, the container system is centrifuged for 10minutes at 2000×g at 0° C.

The volume of the container 71 is divided into a first and secondchamber by pinching the flexible walls of the container 71. The firstchamber comprises the precipitate and a minor part of the plasma. Thecontainer 71 is incubated at 37° C. in order to dissolve the precipitatein the plasma fraction. If needed, an appropriate amount of sterileliquid such as saline may be injected into the container 71 in order tofacilitate the dissolving of the precipitate. If appropriate, thecontainer 71 can be stored until use prior to the incubation step.

EXAMPLE 5

Preparation of compositions comprising tissue repair promotingsubstances prepared as described in Example 4, but with variations inthe ethanol concentration, incubation temperature and pH.

In order to study the effect of the ethanol concentration, theincubation temperature, and the pH during the precipitation, variouspreparations were obtained in a similar manner as described in Example4. Plasma fractions were obtained after centrifugation for 10 minutes at2000×g at 15° C. The plasma fractions were incubated with varyingamounts of 93% w/w ethanol giving concentrations from 6.2% to 9.4% at 0°C. or -3° C., at pH 7.8 or 6.2. pH 6.2 was obtained by the addition of113 mmolar citric acid to the plasma before the ethanol precipitation.The precipitates were centrifuged for 10 minutes at the incubationtemperature of 2000×g.

Subsequent to the isolation of the fraction comprising the precipitate(comprising the tissue repair promoting substances), said precipitatewas incubated at 37° C. for 20 minutes. In four of the experiments, theprecipitate was not completely dissolved and plasma was added to give ahomogeneous solution.

The concentration of fibrinogen was measured as described in Example 1.Results are shown in Table I.

The concentration of the factor XIII was measured by rocketimmunoelectrophoresis according to Laurell's method. Anti-human factorXIII-A rabbit serum was obtained from Diagnostica Stago. A plasma samplewas used as standard. The concentration of factor XIII in the solutionprepared was usually in the order of approximately 20-25 times theconcentration of factor XIII in the plasma of the blood samplescollected.

                  TABLE I                                                         ______________________________________                                                               Addition                                                                      of plas-                                               Ethanol                                                                              Incu-   pH      ma in %                                                                              Fibrinogen                                                                            Recovery                                concen-                                                                              bation  during  of the in the frac-                                                                          of the                                  tration                                                                              temp.   precip- inital tion ob-                                                                              plasma                                  w/w %  °C.                                                                            itation volume tained g/l                                                                            fibrinogen                              ______________________________________                                        6.2    0       7.8     0      41      82                                      9.4    0       7.8     1      36      67                                      6.2    -3      7.8     0      35      70                                      9.4    -3      7.8     1      31      74                                      6.2    0       6.2     2      39      75                                      9.4    0       6.2     2.4    33      89                                      ______________________________________                                    

The fibrinogen concentration in plasma, corrected for the dilution withthe anticoagulant, was 2.6 g/l.

EXAMPLE 6

Preparations as described in Example 5 with further modifications of theethanol concentration and the incubation time

In these experiments the tissue repair promoting substances wereprecipitated with 93% ethanol to give concentrations varying from 7.8%to 10.9% w/w. After incubation at 0° C. for different time intervals,the precipitates were separated and varying amounts of plasma were addedto the precipitates before the incubation at 37° C. After 10 to 15minutes homogeneous solutions were obtained. The concentration offibrinogen was determined as described in Example 1. The concentrationin plasma was corrected so as to compensate for the dilution caused bythe addition of the chemical agents including the anticoagulants. Theresults are shown in Table II.

                                      TABLE II                                    __________________________________________________________________________             Time of                                                                             Addition of                                                                          Fibrinogen concen-                                                                      % Recovery                                             incuba-                                                                             plasma in %                                                                          tration g/l in                                                                          of the                                            Ethanol                                                                            tion, of initial  Fraction                                                                           plasma                                        Donor                                                                             w/w %                                                                              min.  volume Plasma                                                                             obtained                                                                           fibrinogen                                    __________________________________________________________________________    A   7.8  50    5      2.4  18   82                                                9.4  "     5      "    16   82                                                10.9 "     5      "    18   91                                            B   7.8  45    2.5    2.3  19   72                                                9.4  "     2.5    "    20   79                                                10.9 "     2.5    "    20   89                                            C   7.8  120   2.5    2.6  30   83                                                9.4  "     2.5    "    32   89                                                10.9 "     2.5    "    28   95                                            __________________________________________________________________________

EXAMPLE 7

Preparation of a composition containing tissue repair promotingsubstances from blood from a human donor using a sterilized containersystem comprising two containers

For the preparation, a sterilized system corresponding to the systemshown in FIG. 2 was used, with the exception that the syringe 31 wasomitted. Tissue repair promoting substances naturally present in plasmaand platelets were isolated.

The syringe 11 contained agents as described in Example 1.

The collapsible bag 71 was empty.

The human blood was collected as described in Example 1. The system wascentrifuged for 15 minutes at 250×g at 15° C. in order to obtain aplatelet-enriched plasma fraction.

The platelet-enriched plasma fraction was transferred to the container71. The syringe 11 essentially comprising erythrocytes and leucocyteswas disconnected from the container 71.

The container 71 was centrifuged so as to sediment the platelets. Thevolume of the container 71 was divided so as to obtain a first chambercomprising the platelets and a minor part of the plasma and a secondchamber comprising the major part of the plasma.

The container 71 was incubated at -20° C. and the tissue repairpromoting substances derived from plasma and platelets were isolatedwithin the container 71 as described in Example 2.

The resulting solution (1.5 ml) contained 15 g/l of fibrinogen, and therecovery of fibrinogen was 34% of the amount present in the plasmafraction (25 ml containing 2.6 g/l of fibrinogen).

EXAMPLE 8

Preparation of a composition containing tissue repair promotingsubstances naturally present in plasma isolated from human donor blood

The composition was prepared using a sterilized system corresponding tothe one used in Example 4.

The syringe 11 (cf. FIG. 2) contained 6 ml 113 mM tri-sodium citratesolution and 20 IE/ml heparin.

The container 71 contained 6 ml 42% (w/w (NH₄)₂ SO₄ solution.

Human blood was collected as described in Example 1. The system wascentrifuged, and the fraction comprising the major part of the plasmawas transfered to the container 71 as described in Example 4.

After mixing the plasma fraction with the (NH₄)₂ SO₄ solution, thecontainer 71 was centrifuged for ten minutes at 2000×g at 4° C.

The precipitate was separated and dissolved as described in Example 4.70% of the fibrinogen present in the plasma was precipitated by thisprocedure.

EXAMPLE 9

Preparation of a composition containing tissue repair promotingsubstances isolated from the blood of an adult female rabbit and in vivotesting

The composition was prepared using a sterilized system corresponding tothe one shown in FIG. 1. Tissue repair promoting substances present inblood plasma and cells (primarily platelets) were isolated.

The syringe 11 contained 4 ml 113 mM tri-sodium citrate solution and 20IE/ml heparin as anticoagulants and 800 KIE/ml aprotinin as aproteolytic enzyme inhibitor.

The syringe 23 contained adrenalin 0.05 ml 0.1% solution to be used forpromoting growth factor release from platelets.

The syringe 31 contained 2 ml 93% (w/w) ethanol.

26 ml blood was collected from a nembutal anesthetized female rabbitweighing 4 kg by means of the needle 19 inserted in a jugular vein.

After collection of the blood, the system was disconnected from therabbit and the tubing 18 was sealed. The system was centrifuged asdescribed in Example 1.

The volume of the substantially cell-free plasma fraction wasapproximately 22 ml. 20 ml were transferred to the syringe 31 and 2 mlwere used to stir the platelets, leucocytes and a minor fraction oferythrocytes. This cell-containing plasma fraction was transferred tothe syringe 23 as described in Example 1. The syringe 11 containing theerythrocytes was disconnected, and the erythrocytes were resuspended in50 ml sterile saline. This suspension was retransfused to the rabbit.

The composition comprising tissue repair promoting substances derivedfrom plasma and platelets were isolated as described in Example 1.

The volume of the resulting solution was 1.15 ml, and it contained 29g/l of fibrinogen. 87% of the plasma fibrinogen was recovered.

The fibrinogen-containing solution was applied simultaneously with asterile solution containing 1200 NIH/ml thrombin, 100 mM CaCl₂ and 7000KIE/ml aprotinin, during a microsurgical tubal anastomosis procedureperformed on the rabbit from which the blood had been collected withinone hour after collection.

For the application of the two components, the apparatus shown in FIG. 4was used. The apparatus was sterilized prior to use, and keptsterile--externally and internally--during the entire procedure. Thefibronogen-containing solution was transferred to the 3 ml syringe 62and the thrombin-containing solution was transferred to the 1 ml syringe61. The two syringes were placed in the supporting device 66. Theneedles 63 and 64 were connected with the tubing 65 and mounted on thesyringes. During application, the two pistons 67 and 68 were pressedsimultaneously by means of the connective device 69. Thefibrinogen-containing solution and the thrombin solution were mixed inthe proportion 1 to 0.3 at the puncture 70 of the tubing 65.Approximately 10 to 15 seconds after mixing, fibrinogen was converted tofibrin and a stable clot was formed within 3 minutes.

The rabbit was subjected to bilateral microsurgical transection of theistmus, 1 to 3 cm from the corner of the uterus. A 1 cm portion of thetube was excised, followed by end-to-end tubal anastomosis. The proximaland distal segments of the oviduct were assembled by a clamp while thefibrinogen adhesive was applied. Three minutes later the clamp wasremoved. No ordinary suture was used for the anastomosis.

Four weeks after the operation, the rabbit was mated. Immediately beforedelivery it was killed. By dissection, fetuses were observed in bothuterine horns. No adhesion formation was observed.

EXAMPLE 10

Preparation of a composition containing tissue repair promotingsubstances isolated from the blood of a pig.

The composition was prepared in a sterilized system corresponding to theone shown in FIG. 1. Tissue repair promoting substances present in bloodplasma were isolated.

The syringe 11 contained 6 ml 113 mM tri-sodium citrate solution and 20IE/ml heparin as anticoagulants, and 8 mM ε-aminocaproic acid as afibrinolytic inhibitor.

The syringe 23 was empty.

The syringe 31 contained 3 ml ethanol 92% (w/w).

44 ml blood were collected from a nembutal anesthetized pig weighing 32kg by means of the tubing 18 directly inserted in a jugular vein. Aftercollection of the blood, the system was disconnected from the pig andthe tubing 18 was sealed. The system was centrifuged as described inExample 1.

The volume of the substantially cell-free plasma was approximately 33ml. 31 ml were transferred to syringe 31 and 2 ml were transferred tosyringe 23. The syringe 11 essentially comprising the erythrocytes wasdisconnected and discarded.

The tissue repair promoting substances present in plasma were isolatedas described in Example 1. The fibrinogen-containing precipitate wasdissolved in 0.3 ml ethanol-free plasma transferred from syringe 23.

The volume of resulting solution was 2.9 ml, and it contained 27 g/l offibrinogen. 89% of the plasma fibrinogen was recovered.

The solution was stored at -20° C. for three days.

The frozen solution was transferred to 37° C. and liquefaction wasattained within 15 minutes on the day of surgery (performed on the pigfrom which the blood had been collected).

The application was performed as described in Example 9.

In the nembutal anesthetized pig, the liver was exposed by laparatomy.In a lobe an incision of approximately 6 cm in length was cut from themargo towards the central part of the lobe. To reduce bleeding,hemostasis was partly obtained by means of an intestinal clamp. Thewound cleft was assembled simultaneously with the application of thefibrinogen adhesive, and a manual mild pressure was applied forapproximately 3 minutes. After release of the pressure and theintestinal clamp, minor bleeding spots were sealed with the adhesive.

No postoperative complications were observed. One month after theincision there were no macroscopic signs of damage. Histologicalpreparations showed a minor scar of connective tissue--invaginated withblood vessels--approximately three times the size of normal connectivetissue present in pig liver.

I claim:
 1. A method of isolating tissue repair promoting substancesfrom human or animal blood comprising:(a) aseptically collecting a bloodsample from a single human or animal into a sealable sterilizedcontainer system; (b) sealing the container system; (c) preparing tissuerepair promoting substances from the blood sample by processing thesample within the sealed container system, the processing comprising:(i)separating the blood sample into a plurality of fractions including aplasma fraction and a platelet-containing plasma fraction; (ii)precipitating tissue repair promoting substances from the plasmafraction by subjecting the plasma fraction to precipitation-promotingconditions, the precipitation-promoting conditions comprising incubationof the plasma fraction with a precipitation-promoting agent at atemperature at which precipitation occurs, and separating theprecipitate which contains the tissue repair promoting substances fromunprecipitated materials; (iii) subjecting the platelet-containingfraction to conditions which promote the release of tissue repairpromoting substances from the platelets and separating the releasatefrom cells and cell debris; and (iv) combining the precipitate obtainedfrom the plasma fraction with the releasate obtained from theplatelet-containing fraction to form the tissue repair promotingcomposition.
 2. The method of claim 1 wherein theprecipitation-promoting agent is a lower alkanol.
 3. The method of claim2 wherein the lower alkanol is ethanol.
 4. The method of claim 1, 2 or 3wherein the tissue repair promoting substances are precipitated from theplasma fraction at a temperature from -3° C. to 10° C.
 5. The method ofclaim 1 wherein the container system comprises a collection containerfor receiving the blood sample, and the method comprises the additionalstep of introducing an anticoagulant into the collection containerbefore the blood sample is transferred into the collection container. 6.The method of claim 5 comprising the additional step of introducing aproteolytic enzyme inhibitor into the collection container before theprocessing of the blood sample begins.
 7. The method of claim 1 whereinthe release promoting conditions include contacting theplatelet-containing fraction with a substance which promotes growthfactor release from platelets.
 8. The method of claim 7 wherein thesubstance is selected from the group consisting of adenosinediphosphate, adrenalin and collagen.
 9. The method of claim 1 whereinall reagents used in the processing of the blood sample are introducedinto the container system before the blood sample is introduced therein.10. The method of claim 1 wherein more than 60% of the fibrinogenpresent in the blood sample is recovered.
 11. A method of promoting therepair of a tissue of a human or animal comprising:(a) asepticallycollecting a blood sample from a single human or animal into a sealablesterilized container system; (b) sealing the container system (c)preparing tissue repair promoting substances from the blood sample byprocessing the sample within the container system, the processingcomprising:(i) separating the blood sample into a plurality of fractionsincluding a plasma fraction and a platelet-containing plasma fraction;(ii) precipitating tissue repair promoting substances from the plasmafraction by subjecting the plasma fraction to precipitation-promotingpromoting conditions, the precipitation-promoting conditions comprisingincubation of the plasma fraction with a precipitation-promoting agentat a temperature at which precipitation occurs, and separating theprecipitate which contains the tissue repair promoting substances fromunprecipitated materials to obtain an aseptic tissue repair promotingcomposition; (iii) subjecting the platelet-containing fraction toconditions which promote the release of tissue repair promotingsubstances from the platelets and separating the releasate from cellsand cell debris; (iv) combining the precipitate obtained from the plasmafraction with the releasate obtained from the platelet-containingfraction to form the tissue repair promoting composition; and (d)applying an effective amount of the tissue repair promoting compositionto the tissue of the human or animal.
 12. The method of claim 11 whereinthe precipitation-promoting agent is a lower alkanol.
 13. The method ofclaim 12 wherein the lower alkanol is ethanol.
 14. The method of claim11, 45 or 46 wherein the tissue repair promoting substances areprecipitated from the plasma fraction at a temperature from -3° C. to10° C.
 15. The method of claim 11, wherein the container systemcomprises a collection container for receiving the blood sample, and themethod comprises the additional step of introducing an anticoagulantinto the collection container before the blood sample is transferredinto the collection container.
 16. The method of claim 15 comprising theadditional step of introducing a proteolytic enzyme inhibitor into thecollection container before the processing of the blood sample begins.17. The method of claim 11 wherein the release promoting conditionsinclude contacting the platelet-containing fraction with a substancewhich promotes growth factor release from platelets.
 18. The method ofclaim 17 wherein the substance is selected from the group consisting ofadenosine diphosphate, adrenalin and collagen.
 19. The method of claim11 wherein all reagents used in the processing of the blood sample areintroduced into the container system before the blood sample isintroduced therein.
 20. The method of claim 11 wherein more than 60% ofthe fibrinogen present in the blood sample is recovered.
 21. The methodof claim 11 wherein the tissue repair promoting compositions is appliedto the tissue by means of a device comprising a first reservoirassociated with a first piston means which contains the tissue repairpromoting composition and a second reservoir associated with a secondpiston means which contains a substance that catalyzes conversion offibrinogen to fibrin, the first and second piston means of the devicebeing operably linked so that the tissue repair promoting compositionand the catalytic substance are simultaneously expelled and conducted tothe tissue of the human or animal.